1. Field of the Invention
The present invention relates to a method and apparatus for the assessment of the extent of lipid peroxidation in biological and particularly human fluids and suspensions of human tissues. It should be understood however that the present invention can be applicable also to measurement of lipid peroxidation of other substances containing lipids.
2. Description of the Prior Art
Oxygen is required for many life-sustaining metabolic reactions. Acting on an unsaturated fatty acid, active forms of oxygen (free radicals), generally produce lipid peroxides. Oxidation of the unsaturated fatty acid is accompanied by introducing oxygen molecules into double bonds. During the reaction, cis type doubles bonds sites are converted into conjugated double bonds thus producing a hydroperoxide type of lipid peroxide with a conjugated double bond.
When oxygen molecules are directly introduced into saturated or unsaturated fatty acids in a photosensitized oxidation reaction, lipid hydroperoxides with or without a conjugated double bond are generated. In decomposition and polymerization reactions, lipid hydroperoxides as a primary product derived by oxidation, produce secondary oxides of different types.
Oxygen and its activated intermediates may react with cellular components with resultant degradation or inactivation of molecules.
The set of intracellular or extracellular conditions that enable chemical or metabolic generation of reactive oxygen species such as superoxide radicals, hydrogen peroxide, lipid peroxides or related forms is known as oxidative stress. Normally, metabolic activity of the cell is able to control or prevent adverse effects of oxidative stress. The susceptibility to oxidative stress is a function of the overall balance between the factors that exert oxidative stress and those that exhibit antioxidant capability.
Free radical-induced peroxidative damage to membrane lipids has long been regarded as a critical initiating event leading to cell injury. In the presence of a free radical or a free radical initiator, biological materials, and, in particular, cell membranes which contain a relative high proportion of polyunsaturated lipids, become susceptible to oxidation.
The process of lipid peroxidation is thus associated with the loss of membrane polyunsaturated fatty acids and with the formation of hydroperoxides, free radical intermediates and other secondary products. The peroxidation of essential fatty acids may disturb the fine structure of biological membranes and may thus affect the permeability and functions of the membrane. The process of lipid peroxidation, when not aborted, may lead to the rupture of cell membranes and the release of destructive products. As a result, these processes may cause irreversible damage to the cells and may initiate and/or promote the pathogenesis at certain conditions of injury and disease.
Prevention of the potential adverse effects of oxygen and its reactive intermediates is achieved by a number of antioxidant defense systems already presented in the cells or by their enforcement from the outside by virtue of different forms of artificial antioxidants.
Hence providing for a simple, sensitive and reliable method for measuring the extent of lipid peroxidation in biological and particularly human fluids and tissues may constitute an important tool for studying various pathologies ad diseases like for example nutritional imbalance, hereditary diseases, cardiovascular diseases, cancer, diabetes adult respiratory distress syndrome (ARDS) etc.
Decomposition of the peroxides releases energy in the form of chemiluminescence. It is established fact that there is a correlation between the chemiluminescence intensity and the rate of hydroperoxide decomposition. The chemiluminescence can be triggered by heat in the thermochemiluminescence (briefly, TCL) process.
The phenomenon of TCL is mainly caused by two types of basic reactions:
I. Thermal decomposition of dioxatans-cyclic peroxides as demonstrated below:
 greater than Cxe2x95x90O*xe2x86x92 greater than Cxe2x95x90O+hxcexd
II. Oxidation of the lipid radical during heating:
xe2x80x83R+O2xe2x86x92ROOxc2x0
2ROOxc2x0xe2x86x921O2+2Cxe2x95x90O+hxcexd
xe2x80x83where
1O2xe2x80x94singlet oxygen
Cxe2x95x90O*xe2x80x94unstable carbonyl compound
ROOxc2x0xe2x80x94lipid peroxy radical
Both types of reactions exhibits the capability of lipid substances, which may be present in biological fluids and tissues to react via a free-radical oxidation chain reaction to form unstable carbonyl products.
Caused by instability of carbonyl fragments of different origins, low intensity chemiluminescence can be detected as visible light in the range 400-600 nm. Most conventional methods for measuring the lipid peroxidation extent , for example the popular TBA-RS lipid peroxidation test do not rely on chemiluminescence.
In the method described in the U.S. Pat. No. 4,900,680 to Miyazawa et al a sample containing lipids is subjected to a lipid chromatography to separate the lipids into lipid classes, subsequently brought into contact with a luminescent reagent to generate light in an amount corresponding to the content of the lipid hydroperoxide.
The most relevant to the present invention known method is method and apparatus for the measurement of luminescence of biological fluids as described in DE laid open publication No.4421792 to Shnizer et al. herein incorporated by reference. In this document there is described a method for the preparation of a sample of biological fluid which comprises heating the sample under vacuum at a temperature, which is in the range between the freezing point of the sample and the temperature sufficient to induce luminescence emitted by the tested fluid.
Unfortunately the known method does not allow to measure intensity of luminescence in a reliable manner and to establish stable correlation between the intensity of TCL and time. The reason for this lies in the fact that the structure of frozen sample is not continuous and contains voids associated with evacuation of liquid phase. The other reason is relatively slow heating of the sample which did not left enough time for measuring of the TCL.
It can be readily appreciated that the problem of reliable measuring of extent of LPX still needs a solution.
The main object of the present invention is to provide for a method and apparatus for measuring of TCL in which the above mentioned desiderata are sufficiently reduced or overcome.
In particular the main object of the present invention is to provide for a new and improved method and apparatus for measuring the lipid peroxidation extent enabling reliable measuring of TCL and establishing of stable correlation between the intensity of TCL and time.
The above and other objects and advantages of the present invention can be achieved in accordance with the following combination of its essential features, referring to different embodiments thereof.
In an embodiment of the present invention referring to a method of measuring lipid peroxidation in biological fluids, in suspensions of biological tissues or the like, wherein a sample of said fluid or suspension is heated so as to induce therein thermochemical luminescence (TCL) and amount of said TCL can be measured, said method comprising the following sequence of steps:
a) bringing a sample of said fluid or suspension in a receptacle having substantially flat bottom
b) placing said receptacle within a sublimation chamber and putting thereof on a substrate made of substantially metallic material
c) imparting reciprocating motion to said receptacle so as to distribute said sample over the bottom of said receptacle in a substantially homogeneous manner
d) subjecting of said sample residing within said chamber to sublimation at reduced pressure during period of time sufficient for evacuation from said sample of a liquid phase and formation on the bottom of said receptacle of a substantially continuous film consisting of dry solid residual
e) bringing said receptacle with said residual in a processing chamber and placement thereof on a plate having temperature sufficient for inducing TCL and emitting thereof by said residual
f) sensing said TCL and measuring the intensity thereof for example by optical photo-detector during period of time sufficient for establishing stable correlation between TCL intensity and time.
In accordance with one of the preferred embodiments referring to method said receptacle is formed as disposable vessel made of thin metallic foil and said plate is made of aluminum.
As per another preferred embodiment said reciprocating motion is imparted to said sample during at least 40 sec so as to distribute said sample over the bottom of said receptacle as a layer with thickness 0.3-0.5 mm.
According to other preferred embodiment said sublimating step is carried out at reduced pressure not exceeding 2 mbar and during period of time not exceeding 10 min.
In still further preferred embodiment before placement said receptacle onto said plate it is heated up to at least 60 deg. C.
And in yet another preferred embodiment relating to method said TCL intensity is measured in the range of 400-600 nm during at least 300 sec.
According to the first preferred embodiment referring to an apparatus for measuring lipid peroxidation in biological fluids in suspensions of biological tissues or the like, wherein a sample of said fluid or suspension is heated so as to induce therein thermochemical luminescence (TCL) and amount of said TCL can be measured, said apparatus comprising:
a) sublimation chamber provided with a means for homogeneous distributing of said sample within a receptacle and with a sublimating means capable to evacuate liquid phase from said sample so as to form a thin substantially continuous film consisting of dry solid residual, said means for homogeneous distributing comprises a substrate made of preferably metallic material, said substrate is provided with a depression for receiving said receptacle therein and there is provided a driving means for imparting reciprocating motion to said substrate
b) heating chamber provided with a plate for placing said receptacle thereon, with a heating means for heating said plate and with a means for detection and measuring said CTL
c) data recording unit for presentation in visual manner results of measuring of said TCL
d) control and data processing unit for controlling said means for homogeneous distributing, said driving means, said sublimating means, said heating means, said means for detection and measuring said TCL and said data recording unit.
According to one of the preferred embodiments of the apparatus said sublimating means comprises vacuum pump capable to generate reduced pressure within said sublimation chamber.
In accordance with the other preferred embodiment said substrate is formed as a disc made of aluminum, said disc is supported by elastic tubular elements connecting said disc with the basis of said sublimating chamber and said driving means comprises a motor imparting reciprocating motion to said plate via a cam.
And as per still further preferred embodiment of the apparatus said heating means comprises transistor and temperature sensor and said means for detection and measuring TCL comprises photo-electronic multiplier.
The present invention in its various embodiments has only been summarized briefly. For better understanding of the present invention as well of its advantages, reference will now be made to the following description of its embodiments.